Taichi Q. Itoh1 and Akira Matsumoto21Graduate School of Systems Life Sciences, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan and Research Fellow of the Japan Society for the Promotion of Science, Japan and 2Department of Biology, Juntendo University School of Medicine, 1-1 Hiraga Gakuendai, Inzai, Chiba 270-1695, JapanDOI: 10.2144/000113781

Abstract

Traditional cloning procedures with restriction enzymes have limitations and lack efficiency when constructing recombinant molecules using multiple fragments. SA-cloning can construct an expression plasmid over 10kb with multiple fragments at one time by the self-assembly of complementary staggered overhangs on PCR-amplified fragments.

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⇒ATTENTION:

*HINT:

✋REST

Procedure

⇒ATTENTION:The following procedure is a typical example of SA-cloning to insert one fragment into a vector via short sequences. You can apply this procedure to multiple fragment assembly or in-frame seamless cloning with appropriate primer sets.

Amplification of the target gene and cloning vector

In SA-cloning, two PCR reactions should be prepared for one insert (Fig. 1, Tubes A and B); one contains a tailed forward primer (Fig. 1, primer 1') and an untailed gene specific reverse primer (Fig. 1, primer 2) and the other has an untailed gene specific forward primer (Fig. 1, primer 1') and a tailed reverse primer (Fig. 1, primer 2'). Additionally, two PCR reactions should be prepared for the vector (Fig. 1, Tube C and D).

Denaturation and re-annealing are performed in thin-wall PCR tubes (Fig. 1, Tube G and H) to produce fragments with single-stranded overhangs, one tailed on the 5' end and the other on the 3' end, using 500 ng of pooled PCR fragments. The self-assembly of fragments to make a desired circular plasmid is also performed in thin-wall PCR tubes (Fig. 1, Tube I).

20. Spread the full volume on a pre-warmed selective plate with appropriate antibiotics.

✋RESTIncubate overnight at 37°C.

21. Pick up several colonies and check the vector sequence by PCR or sequencing.

*HINT:Instead of One Shot® TOP10 Chemically Competent E. coli, other competent cells can be used.

⇒ATTENTION:Almost all colonies usually have a positive plasmid as far as our experience goes, however we strongly recommend that you check several colonies with PCR and/or sequencing analysis.

Troubleshooting

Non-specific bands in PCR

Raise temperature at annealing step in PCR.

Reduce the amount of a template DNA.

Re-design the overhang sequences.

Prepare a new primer that anneals to a different site.

If there is no improvement with the changes described above, purify the desired fragment after gel electrophoresis.

Negative Colonies

Be sure to check the quality and quantity of the PCR reaction in Tube A, B, C and D by gel electrophoresis.

Be sure to troubleshoot as described above for any non-specific bands seen in the gel following PCR.

For undigested template DNA, add 1µL of DpnI into the reaction and completely digest it.

Do not rapidly cool down the reaction after the denaturation step.

Check whether the sequences of overhang regions are unique. Although you need to design overhang regions that are similar to each other, for example to establish a repetitive sequence, we strongly recommend that the sequence homology between overhang regions be less than 75%.

Lengthen the overhang region. The minimum length of the overhang region for successful self-assembly is six base pairs.

No Colonies

Check the quality and quantity of the PCR reaction in Tube A, B, C and D by gel electrophoresis. See above.

Increase the concentration of PCR fragments in the denaturation and re-annealing steps (Fig. 1 Tube G and H).

Check whether each pair of overhang sequences is perfectly complementary. Even a single mismatch in an overhang region drastically reduces the cloning efficiency in SA-cloning.

Lengthen the overhang region. The minimum length of overhangs for successful self-assembly is six base pairs.